Accurate chromosome segregation depends on chromosome condensation, the establishment and maintenance of the linkage between sister chromatids, and the rapid and synchronous separation of sister chromatids at the metaphase-anaphase transition. Defects in any of these process leads to errors in chromosome segregation that can lead to genetic instability and aneuploidy that are characteristic of tumorogenesis and birth defects. This proposal describes experiments in budding yeast to investigate the mechanisms regulating these fundamental aspects of chromosome behavior. Scc1p/Mcd1p is required for condensation, establishment and maintenance of sister chromatid linkage, and its dissociation from chromosomes s necessary for sister chromatid separation. To elucidate the role of Scc1p in these processes it is critical to identify proteins that contribute to sister chromatid separation. Using a green fluorescent protein (GFP)-based sister separation assay, a group of conditional loc (loss of cohesion) mutants were identified that failed to separate sister chromatids at the metaphase-anaphase transition. The combined strengths of genetic analysis, GFP-tagged chromosomes and biochemistry will be utilized to investigate the role of Scc1p and the proteins defined by these loc mutants in sister chromatid condensation, cohesion and separation. Chromosome condensation in living cells will be monitored by quantifying the distance between five LacO arrays integrated approximately 200 kb apart on chromosome IV and five similarly spaced TetO arrays in the rDNA locus. These arrays will be visualized by binding of a GFP-Lac repressor (GFP- LacI) fusion protein and yellow fluorescent protein-Tet repressor (YFP-TetR) fusion protein, respectively. This novel technique will be used to determine the kinetics